Hybrid plants have been demonstrated to be superior to inbred lines with respect to yield and vigor. The production of hybrid seed on a large scale is challenging because many crops have both male and female reproductive organs (stamen and pistil) on the same plant, either within a single flower or in separate flowers. This arrangement results in a high level of self pollination and makes large scale directed crosses between inbred lines difficult to accomplish.
To guarantee that outcrossing will occur during the production of hybrid seed, breeders have either manually or mechanically removed stamens from one parental line, or exploited male sterility mutations that disrupt pollen development. Manual emasculation is labor intensive and impractical for plants with small bi-sexual flowers.
Important crops like rice, corn, wheat are self-pollinating plants, and therefore there is a need to develop systems for pollination control to assist in the production of F1 hybrids. All currently available systems are based on the introduction of a male sterility trait to one parental plant followed by the introduction of a fertility-restorer gene, as a result of cross-pollination, to produce fertile hybrid plants.
Male reproductive processes in flowering plants occur in the anther. This organ is composed of several tissues and cell types, and is responsible for producing pollen grains that contain the sperm cells. A specialized anther tissue, the tapetum, plays an important role in pollen formation. The tapetum surrounds the pollen sac early in anther development, degenerates during the later stages of development and is not present as an organized tissue in the mature anther. The tapetum produces a number of proteins and other substances that either aid in pollen development or become components of the pollen outer wall. It is known that many male sterility mutations interfere with tapetum cell differentiation and/or function. Thus tapetal tissue is believed to be essential for the production of functional pollen grains.
Currently available male-sterile-based pollination systems are not satisfactory. In many cases, male sterility is unstable under different climatic conditions (Fan and Stefansson, 1986) and partial female sterility of the male-sterile plants and/or reversion to fertility of male-sterile plants has been observed. Furthermore, the restorer genes used to restore fertility to the male sterile lines have also been found to be unstable. In some cases, prominent morphological changes in the male-sterile flowers were evident (Denis et al., 1993).
Another approach to the control of fertility is based on the use of a cytoplasmic-male-sterility system. See, for example, Patterson, U.S. Pat. No. 3,861,079. However, reliance on a single cytoplasmic-male-sterile system for the production of all hybrid plants is undesirable because it leaves the entire hybrid stock vulnerable to plant pathogens. For example, extensive use of one corn cytotype, cmsT lead to an eptiphytic outbreak of Southern Corn Leaf Blight in the early 1970's. Thus, it is important to develop alternative methods to produce male sterile lines in plant species where only a single male-sterility system is available.
The present invention relates to a new method for making hybrid plants via a novel male-sterility/restorer system. The method incorporates into one plant line (line A) a male sterility system (an anther-specific promoter driving a suicidal gene) linked to a phenotypic trait (herbicide-resistance, seed coat color, seed plumpness, etc.). The phenotypic trait allows one to identify and maintain the male sterile progeny. Furthermore the male sterility system is flanked by target sites (recombination sequences) of a site-specific recombinase such that the introduction of recombinase activity excises the male sterility system, thus restoring fertility to the progeny of the male sterile plant. Plant line B is generated by incorporating a gene encoding a site-specific recombinase under the control of a constitutive (e.g. ubiquitin, CaMV35S) or cell-specific (e.g. anther, tapetum, zygote, embryo, etc.) promoter. The site-specific recombinase recognizes and interacts with the site-specific recombination sequences present in line A. After multiplication of lines A and B, the two plant lines are crossed, and expression of the site-specific recombinase in the zygote or early embryonic cells results in the recombinase enzyme excising all of the DNA between the target sites. In accordance with one embodiment the sterility system and the linked marker system are excised, giving rise to fertile plants that produce hybrid seeds.
This method can be generally applied to plants for which the pollination control systems have not yet been established, or with which the production of hybrids is not efficient. Accordingly, the present invention provides recombinant expression vectors and a method for producing the male sterile and fertility restorer plant lines utilized to produce fertile F1 hybrid plant entities.